Unveiling the multifaceted mechanisms of action in nonionic and cationic biocide combinations against Gram-negative...

Quaternary ammonium compounds (QACs) combined with nonionic surfactants have been among the most effective disinfectants for over half a century, leveraging QACs' broad-spectrum antimicrobial activity that targets microbial membranes. However, the specific interactions between QACs and microbial membranes, as well as the role of nonionic surfactants in disinfection, remain unclear. This study investigates these mechanisms using two representative surfactants: the cationic didecyldimethyl ammonium chloride (DDAC) and the nonionic hexaethylene glycol monododecyl ether (C₁₂E₆). The antimicrobial activity of these agents, individually and sequentially, was assessed against Gram-negative bacteria through a series of in vitro assays, including outer membrane (OM) permeability, inner membrane (IM) depolarization, and live/dead bacterial imaging. Further insights into membrane interactions were obtained using model lipid bilayers in conjunction with antimicrobial efficacy matrices, FICI (fractional inhibition concentration index), fluorescent liposome leakage, small-angle neutron scattering (SANS), and neutron reflectivity (NR). Results indicate that C₁₂E₆ binds to the rough A lipopolysaccharide (RaLPS) head region in the OM, reassembling it into heterogeneous aggregates but with limited penetration to cause IM disruption. Conversely, DDAC induced structural disruptions in both OM and IM, resulting in low inhibitory concentrations and rapid bacterial killing. In mixtures, the C₁₂E₆ : DDAC ratio significantly influences antimicrobial efficacy, with higher C₁₂E₆ levels inhibiting DDAC's effective membrane interactions.